PROJECT SUMMARY Dental pulp tissue exposed to a mechanical trauma or cariogenic process can result in root canal and/or periapical infections, which can be treated via an endodontic procedure. A regenerative endodontic procedure (REP) attempts to revitalize pulp-dentin tissue from a previously necrotic or inflamed pulp, thus, allowing for continued development of the pulp-dentin structure, particularly for a young immature root. However, the current REP has resulted in unfavorable outcomes, including tooth discoloration, cervical root fractures, inadequate pulp-dentin tissue structure formation, and multiple clinic visits. In order to tackle the challenges of the current REP, a highly interdisciplinary and innovative strategy is proposed for efficient root canal disinfection and pulp-dentin tissue revitalization using an antibiotics and nitric oxide (NO) releasing biomimetic nanomatrix gel. We hypothesize that the antibiotics and NO releasing nanomatrix gel will demonstrate an efficient antibacterial effect and recruit endogenous stem cells to induce pulp-dentin revitalization, while providing a favorable pulp-dentin tissue mimicking extracellular matrix environment. The proposed strategy has several advantages over the current REP: 1) preventing tooth discolorations by removal of minocycline, 2) preventing cervical root fractures by avoidance of calcium hydroxide, 3) antibacterial effect of the NO, 4) sustained NO release from the nanomatrix gel by enzyme mediated degradation, 5) reducing inflammatory responses by avoidance of reopening root canals and stimulated bleeding, 6) biocompatibility and biodegradability of the nanomatrix gel as a functional extracellular matrix, and 7) less extensive pulp access opening, clinical visits, and restorations with cost-effective materials such as composites resin. Therefore, three specific aims are proposed to evaluate the hypothesis. Specific Aim 1 is to evaluate the anti-biofilm effects of the antibiotics and NO releasing biomimetic nanomatrix gel in clinical samples from the endodontic infection. Specific Aim 2 is to evaluate the cellular effects of the antibiotics and NO releasing biomimetic nanomatrix gel on dental pulp stem cells. Specific Aim 3 is to develop a beagle infected tooth model and evaluate the revitalization capacity of the antibiotics and NO releasing biomimetic nanomatrix gel. The outcomes from this proposal will demonstrate the anti-biofilm effect and revitalization potential of the antibiotics and NO releasing nanomatrix gel for the treatment of endodontic infections. Further, the outcomes will also lead to future clinical studies for a young immature root as well as a mature root canal infection with traumatic exposure and bacterial associated cases.